Bottom Line:
Transcription factors and chromatin-remodeling complexes are key determinants of embryonic stem cell (ESC) identity.BRD4 maintains transcription of core stem cell genes such as OCT4 and PRDM14 by occupying their super-enhancers (SEs), large clusters of regulatory elements, and recruiting to them Mediator and CDK9, the catalytic subunit of the positive transcription elongation factor b (P-TEFb), to allow Pol-II-dependent productive elongation.Our study describes a mechanism of regulation of ESC identity that could be applied to improve the efficiency of ESC differentiation.

Figure 3: BET Inhibition and BRD4 Suppression Lead to EMT Induction and Neuroectodermal Lineage Commitment of ESCs(A) Downregulation of the epithelial marker E-cadherin (ECAD) in FGF-, vehicle-, or compound-treated hESCs, as detected by immunofluorescence analysis after 5 days of compound treatment. The scale bar represents 100 μm.(B) Expression over time of a panel of EMT, neuroectodermal, and mesendodermal markers in hESCs following BET inhibition, as detected by qRT-PCR. Dashed line represents expression in vehicle-treated cells.(C) Gene set enrichment analysis of upregulated genes following 5 days of compound treatment in Gene Ontology terms compared to FGF-treated hESCs.(D) Expression of EMT (red) and mesendodermal (green) genes following BRD4 depletion in hESCs, as detected by qRT-PCR. Dashed line represents NTC-transduced hESCs.(E) Representative images of teratomas derived from NTC and shBrd4-transduced mESCs. The scale bar represents 1 cm.(F) Weight distribution of NTC- and Brd4-depleted mESC-derived tumors. Horizontal line represents the median value.(G) Representative micrographs of hematoxylin and eosin sections of NTC or shBrd4-mESCs-derived tumors. EC, ectoderm; EN, endoderm; M, mesoderm.(H) Expression of Ki67 and NeuN in shBrd4-derived tumors compared to NTC-derived teratomas as detected by immunohistochemistry. The scale bar represents 200 μm.(I) Oct4 expression levels following compound treatment in mESCs transduced with an empty vector or Oct4 cDNA-expressing vector, as detected by qRT-PCR.(J) Expression by qRT-PCR of EMT and neuroectodermal genes (Ncad and Nestin) following BET inhibition in control and Oct4-expressing mESCs.(K) Expression by qRT-PCR of mesendodermal genes (Brachyury and Cdx2) following BET inhibition in control and Oct4-expressing mESCs.Error bars represent SD. *p ≤ 0.05. See also Table S3 and Figures S4 and S5.

Mentions:
We next investigated whether BRD4 inhibition results in ESC differentiation together with suppression of stem cell genes and loss of self-renewal capacity. Compound treatment of ESCs modulated the expression of genes involved in epithelialto-mesenchymal transition (EMT) (Figures 3A and 3B), a defining feature of ESCs undergoing differentiation. Accompanying the emergence of an EMT signature, we observed an enrichment of neuroectodermal differentiation markers (e.g., NES, NCAD, and SOX1) relative to markers of other lineages in both hESCs (Figures 3B and 3C) and mESCs (Figures S4A and S4B), indicating lineage specificity associated with BET inhibition. Consistently, individual silencing of BRD4 mimicked the preferential regulation of EMT and neuroectodermal markers over other line-ages observed following pharmacologic BET inhibition in human (Figure 3D) and mouse ESCs (Figure S4C). BET inhibitor treat ment of ESCs significantly affected both the number and morphology of embryoid bodies (EBs) (Figures S5A and S5B), indicating that BRD4 activity is required for the formation of tridimensionally organized EBs. Moreover, BET inhibition resulted in suppression of stem cell genes (Figure S5C) and induction of neuroectodermal lineage genes over genes of other lineages in EBs formation assay (Figure S5D), highlighting the specific line-age commitment following BET inhibition.

Figure 3: BET Inhibition and BRD4 Suppression Lead to EMT Induction and Neuroectodermal Lineage Commitment of ESCs(A) Downregulation of the epithelial marker E-cadherin (ECAD) in FGF-, vehicle-, or compound-treated hESCs, as detected by immunofluorescence analysis after 5 days of compound treatment. The scale bar represents 100 μm.(B) Expression over time of a panel of EMT, neuroectodermal, and mesendodermal markers in hESCs following BET inhibition, as detected by qRT-PCR. Dashed line represents expression in vehicle-treated cells.(C) Gene set enrichment analysis of upregulated genes following 5 days of compound treatment in Gene Ontology terms compared to FGF-treated hESCs.(D) Expression of EMT (red) and mesendodermal (green) genes following BRD4 depletion in hESCs, as detected by qRT-PCR. Dashed line represents NTC-transduced hESCs.(E) Representative images of teratomas derived from NTC and shBrd4-transduced mESCs. The scale bar represents 1 cm.(F) Weight distribution of NTC- and Brd4-depleted mESC-derived tumors. Horizontal line represents the median value.(G) Representative micrographs of hematoxylin and eosin sections of NTC or shBrd4-mESCs-derived tumors. EC, ectoderm; EN, endoderm; M, mesoderm.(H) Expression of Ki67 and NeuN in shBrd4-derived tumors compared to NTC-derived teratomas as detected by immunohistochemistry. The scale bar represents 200 μm.(I) Oct4 expression levels following compound treatment in mESCs transduced with an empty vector or Oct4 cDNA-expressing vector, as detected by qRT-PCR.(J) Expression by qRT-PCR of EMT and neuroectodermal genes (Ncad and Nestin) following BET inhibition in control and Oct4-expressing mESCs.(K) Expression by qRT-PCR of mesendodermal genes (Brachyury and Cdx2) following BET inhibition in control and Oct4-expressing mESCs.Error bars represent SD. *p ≤ 0.05. See also Table S3 and Figures S4 and S5.

Mentions:
We next investigated whether BRD4 inhibition results in ESC differentiation together with suppression of stem cell genes and loss of self-renewal capacity. Compound treatment of ESCs modulated the expression of genes involved in epithelialto-mesenchymal transition (EMT) (Figures 3A and 3B), a defining feature of ESCs undergoing differentiation. Accompanying the emergence of an EMT signature, we observed an enrichment of neuroectodermal differentiation markers (e.g., NES, NCAD, and SOX1) relative to markers of other lineages in both hESCs (Figures 3B and 3C) and mESCs (Figures S4A and S4B), indicating lineage specificity associated with BET inhibition. Consistently, individual silencing of BRD4 mimicked the preferential regulation of EMT and neuroectodermal markers over other line-ages observed following pharmacologic BET inhibition in human (Figure 3D) and mouse ESCs (Figure S4C). BET inhibitor treat ment of ESCs significantly affected both the number and morphology of embryoid bodies (EBs) (Figures S5A and S5B), indicating that BRD4 activity is required for the formation of tridimensionally organized EBs. Moreover, BET inhibition resulted in suppression of stem cell genes (Figure S5C) and induction of neuroectodermal lineage genes over genes of other lineages in EBs formation assay (Figure S5D), highlighting the specific line-age commitment following BET inhibition.

Bottom Line:
Transcription factors and chromatin-remodeling complexes are key determinants of embryonic stem cell (ESC) identity.BRD4 maintains transcription of core stem cell genes such as OCT4 and PRDM14 by occupying their super-enhancers (SEs), large clusters of regulatory elements, and recruiting to them Mediator and CDK9, the catalytic subunit of the positive transcription elongation factor b (P-TEFb), to allow Pol-II-dependent productive elongation.Our study describes a mechanism of regulation of ESC identity that could be applied to improve the efficiency of ESC differentiation.